Mycobacteria employ several mechanisms to exploit the host immune system for its own benefit. Rather than being killed by macrophages, specialized immune cells that normally engulf and eradicate pathogens, mycobacteria survive and persist by using macrophages as their host niche. The infected macrophages then recruit additional macrophages and other immune cells to the infection foci to form a granuloma, an organized aggregate of immune cells that is a hallmark of tuberculous infections. Although traditionally thought to be a host-protective structure, recent evidence from the Mycobacterium marinum- zebrafish model of infection has demonstrated that mycobacteria exploit the developing granuloma as a means to disseminate infection. It does so by secreting the bacterial factor ESAT-6 which induces the expression of host matrix metalloproteinase-9 (MMP9) from epithelial cells. MMP9 is a member of a larger family of proteases that modify the extracellular matrix, as well as cell migration and inflammatory molecules. During mycobacterial infections, MMP9 is required for the recruitment of macrophages to the developing granuloma. This proposal will investigate the mechanism by which ESAT-6 induces MMP9 secretion to promote granuloma development. I will begin by determining if ESAT-6 is necessary for the activation of mmp9 expression or if additional bacterial secreted factors play a role. I will take advantage of the optical transparency of zebrafish embryos to investigate if ESAT-6 is sufficient to induce macrophage recruitment, as infection may be required for granuloma formation. I hypothesize that ESAT-6 interacts directly with epithelial cells to induce mmp9 expression and will investigate this in vitro using human epithelial cell lines. I will identify the host determinant that binds to ESAT- 6 and transmits the granuloma-promoting signal. Lastly, I will employ mutational analysis to determine which regions of ESAT-6 are required for mmp9 expression. The experiments outlined in this proposal will shed light on the mechanisms employed by mycobacteria to exploit the host immune system and promote pathogenesis. PUBLIC HEALTH RELEVANCE: Due to the increasing prevalence of multi-drug resistant strains of Mycobacterium tuberculosis, coupled with the HIV pandemic, human tuberculosis disease remains a major global health problem. This proposal will investigate the interactions between host and pathogen factors during mycobacterial infections. These experiments may lead to the discovery and characterization of new host drug targets and reduce the time required to treat drug-resistant tuberculosis infections. )